The research described herein will investigate patterns of stimulation of phagocytic leukocytes and determine to what extent common pathways are following during various stimulatory conditions. It is our goal to determine the interrelatedness of the various indicators of leukocyte stimulation. We will employ synergistic conditions (i.e., very low levels of stimuli in combination) which may be more reflective of physiological conditions in vivo than the use of saturating levels of stimuli to probe these activation mechanisms. Retinoids, which we have shown to have novel stimulatory properties, will also be used as tools to study leukocyte stimulation mechanisms. Osteocalcin, a major non-collagenous bone protein which is chemotactic for monocytes, will be utilized as a stimulus. We will determine what other features of leukocyte biology are stimulated by osteocalcin and investigate the mechanisms of action of this molecule. We will employ fluorescence video imaging microscopy to monitor stimulus-induced changes in cytoplasmic pH and pCa++ in leukocytes and to follow the intracellular processing of osteocalcin in target cells. These experiments will utilize biochemical, cell biological, and morphological techniques to explore the mechanisms involved in leukocyte stimulation. A synthesis of these approaches, and the use of several stimulatory conditions, should provide a deeper understanding of this complex process. A better understanding of leukocyte function and the mechanisms which control these cells may lead to clinical applications which are important therapeutically.